The portal protein plays essential roles at different steps of the SPP1 DNA packaging process

AbstractA large number of viruses use a specialized portal for entry of DNA to the viral capsid and for its polarized exit at the beginning of infection. These families of viruses assemble an icosahedral procapsid containing a portal protein oligomer in one of its 12 vertices. The viral ATPase (terminase) interacts with the portal vertex to form a powerful molecular motor that translocates DNA to the procapsid interior against a steep concentration gradient. The portal protein is an essential component of this DNA packaging machine. Characterization of single amino acid substitutions in the portal protein gp6 of bacteriophage SPP1 that block DNA packaging identified sequential steps in the packaging mechanism that require its action. Gp6 is essential at early steps of DNA packaging and for DNA translocation to the capsid interior, it affects the efficiency of DNA packaging, it is a central component of the headful sensor that determines the size of the packaged DNA molecule, and is essential for closure of the portal pore by the head completion proteins to prevent exit of the DNA encapsidated. Functional regions of gp6 necessary at each step are identified within its primary structure. The similarity between the architecture of portal oligomers and between the DNA packaging strategies of viruses using portals strongly suggests that the portal protein plays the same roles in a large number of viruses

Overview of Clostridium difficile Infection: Life Cycle, Epidemiology, Antimicrobial Resistance and Treatment

The use of antimicrobial agents and acquired resistances explains in part the emergence and spreading of epidemic strains of Clostridium difficile. Continued use of antimicrobial therapy still represents an acute danger in triggering the emergence and spreading of new resistant and multiresistant strains including against first-line antibiotics. We examine the pathway of peptidoglycan synthesis in this organism and associated resistances, as well as resistance to other classes of antibiotics. The life cycle of C. difficile involves growth, spore formation and germination. Spores endow the organism with a formidable capacity of persistence in the environment and in the host, resistance, dissemination and infectious potential. Highly resistant spores produced by antibiotic-resistant/multiresistant strains may be one of the most serious challenges we face in what concerns the containment of C. difficile. Finally, we review recent developments in the treatment and prevention of C. difficile infection

Rethinking the Niche of Upper-Atmosphere Bacteria: Draft Genome Sequences of Bacillus aryabhattai C765 and Bacillus aerophilus C772, Isolated from Rice Fields

Here, we report two genome sequences of endospore-forming bacteria isolated from the rice fields of Comporta, Portugal, identified as Bacillus aryabhattai C765 and Bacillus aerophilus C772. Both species were previously identified in air samples from the upper atmosphere, but our findings suggest their presence in a wider range of environmental niches.FCT grant: (PEST-E/EQB/LA0004/2011), FCT contracts: (IF/00268/2013/CP1173/CT00061, SFRH/BPD/89907/2012)

Characterization of Clostridium difficile 027 strains from an outbreak in a Portuguese hospital

C. difficile infection (CDI) is the cause of an intestinal disease mediated by two potent cytotoxins, TcdA and TcdB. Symptoms of CDI can range from asymptomatic colonization or mild diarrhea, to life-threatening inflammatory lesions such as pseudomembraneous colitis, toxic megacolon or bowel perforation. In part because of the recent emergence of so-called hypervirulent strains, especially (but not exclusively) those belonging to ribotype 027, C. difficile is now considered a main nosocomial enteric pathogen. Hypervirulent epidemic strains have been associated with more severe disease conditions, with higher relapse rates and increased mortality. Health care-associated CDI develops in hospitalized patients undergoing antibiotic treatment because C. difficile can colonize the gut if the normal intestinal microbiota is disturbed. However, C. difficile is also emerging as an important pathogen in the community, as well as in animal husbandry. The organism is an obligate anaerobe, and has the ability to form spores. Spores are extremely resilient and can accumulate and remain viable in the environment or in the host for long periods of time. Spores that remain latent in the gut are responsible for the recurrence of C. difficile-associated disease (CDAD) when antibiotic therapy is stopped. At least some of the hypervirulent epidemic strains show a greater sporulation capacity in vitro, as well as robust toxin production. The first detection of C. difficile 027 hypervirulent epidemic strains implicated in a hospital outbreak in Portugal dates from January 2012, involving 12 patients, with a crude mortality rate of 50%. Here we report on the genetic characterization of those strains as well as the antibiotic resistance profile, toxin production, and rate and efficiency of spore formation. In parallel, C. difficile 027 non-outbreak strains isolated from other Portuguese health care facilities are also investigated

Genetic Competence Drives Genome Diversity in Bacillus subtilis

This deposit is composed by the main article plus the supplementary materials of the publication.Prokaryote genomes are the result of a dynamic flux of genes, with increases achieved via horizontal gene transfer and reductions occurring through gene loss. The ecological and selective forces that drive this genomic flexibility vary across species. Bacillus subtilis is a naturally competent bacterium that occupies various environments, including plant-associated, soil, and marine niches, and the gut of both invertebrates and vertebrates. Here, we quantify the genomic diversity of B. subtilis and infer the genome dynamics that explain the high genetic and phenotypic diversity observed. Phylogenomic and comparative genomic analyses of 42 B. subtilis genomes uncover a remarkable genome diversity that translates into a core genome of 1,659 genes and an asymptotic pangenome growth rate of 57 new genes per new genome added. This diversity is due to a large proportion of low-frequency genes that are acquired from closely related species. We find no gene-loss bias among wild isolates, which explains why the cloud genome, 43% of the species pangenome, represents only a small proportion of each genome. We show that B. subtilis can acquire xenologous copies of core genes that propagate laterally among strains within a niche. While not excluding the contributions of other mechanisms, our results strongly suggest a process of gene acquisition that is largely driven by competence, where the long-term maintenance of acquired genes depends on local and global fitness effects. This competence-driven genomic diversity provides B. subtilis with its generalist character, enabling it to occupy a wide range of ecological niches and cycle through them.Fundação para a Ciência e a Tecnologia grants: (PTDC/EBB-BIO/119006/2010, PEst-OE/EQB/LA0004/2011, SFRH/BPD/89907/2012, SFRH/BD/29397/06); FEDER grant: (LISBOA-01-0145-FEDER-007660).info:eu-repo/semantics/publishedVersio

Novel Secretion Apparatus Maintains Spore Integrity and Developmental Gene Expression in Bacillus subtilis

Sporulation in Bacillus subtilis involves two cells that follow separate but coordinately regulated developmental programs. Late in sporulation, the developing spore (the forespore) resides within a mother cell. The regulation of the forespore transcription factor σG that acts at this stage has remained enigmatic. σG activity requires eight mother-cell proteins encoded in the spoIIIA operon and the forespore protein SpoIIQ. Several of the SpoIIIA proteins share similarity with components of specialized secretion systems. One of them resembles a secretion ATPase and we demonstrate that the ATPase motifs are required for σG activity. We further show that the SpoIIIA proteins and SpoIIQ reside in a multimeric complex that spans the two membranes surrounding the forespore. Finally, we have discovered that these proteins are all required to maintain forespore integrity. In their absence, the forespore develops large invaginations and collapses. Importantly, maintenance of forespore integrity does not require σG. These results support a model in which the SpoIIIA-SpoIIQ proteins form a novel secretion apparatus that allows the mother cell to nurture the forespore, thereby maintaining forespore physiology and σG activity during spore maturation

Clostridioides difficile para-Cresol Production Is Induced by the Precursor para-Hydroxyphenylacetate.

Clostridioides difficile is an etiological agent for antibiotic-associated diarrheal disease. C. difficile produces a phenolic compound, para-cresol, which selectively targets gammaproteobacteria in the gut, facilitating dysbiosis. C. difficile decarboxylates para-hydroxyphenylacetate (p-HPA) to produce p-cresol by the action of the HpdBCA decarboxylase encoded by the hpdBCA operon. Here, we investigate regulation of the hpdBCA operon and directly compare three independent reporter systems; SNAP-tag, glucuronidase gusA, and alkaline phosphatase phoZ reporters to detect basal and inducible expression. We show that expression of hpdBCA is upregulated in response to elevated p-HPA. In silico analysis identified three putative promoters upstream of hpdBCA operon-P1, P2, and P?54; only the P1 promoter was responsible for both basal and p-HPA-inducible expression of hpdBCA We demonstrated that turnover of tyrosine, a precursor for p-HPA, is insufficient to induce expression of the hpdBCA operon above basal levels because it is inefficiently converted to p-HPA in minimal media. We show that induction of the hpdBCA operon in response to p-HPA occurs in a dose-dependent manner. We also identified an inverted palindromic repeat (AAAAAG-N13-CTTTTT) upstream of the hpdBCA start codon (ATG) that is essential for inducing transcription of the hpdBCA operon in response to p-HPA, which drives the production of p-cresol. This provides insights into the regulatory control of p-cresol production, which affords a competitive advantage for C. difficile over other intestinal bacteria, promoting dysbiosis.IMPORTANCE Clostridioides difficile infection results from antibiotic-associated dysbiosis. para-Cresol, a phenolic compound produced by C. difficile, selectively targets gammaproteobacteria in the gut, facilitating dysbiosis. Here, we demonstrate that expression of the hpdBCA operon, encoding the HpdBCA decarboxylase which converts p-HPA to p-cresol, is upregulated in response to elevated exogenous p-HPA, with induction occurring between >0.1 and ?0.25?mg/ml. We determined a single promoter and an inverted palindromic repeat responsible for basal and p-HPA-inducible hpdBCA expression. We identified turnover of tyrosine, a p-HPA precursor, does not induce hpdBCA expression above basal level, indicating that exogenous p-HPA was required for p-cresol production. Identifying regulatory controls of p-cresol production will provide novel therapeutic targets to prevent p-cresol production, reducing C. difficile's competitive advantage

Resistance of Clostridium difficile from ribotype 017 to imipenem: contribution of the whole genome sequencing

A infeção por Clostridium difficile é a principal causa de diarreia infeciosa associada aos cuidados de saúde. Neste estudo, caracterizámos um conjunto de estirpes clínicas de Clostridium difficile, provenientes de diversos hospitais portugueses, com o objetivo de estudar a resistência aos carbapenemos neste agente patogénico. Um total de 191 estirpes clínicas, isoladas entre 2012 e 2015 de 15 hospitais em Portugal, foram incluídas no estudo; a suscetibilidade ao imipenemo foi determinada por um método de gradiente de difusão em agar. Foram selecionadas estirpes sensíveis e resistentes ao imipenemo, para estudos fenotípicos adicionais e para contributo da sequenciação do genoma completo. A resistência ao imipenemo foi detetada em 24 (12,6%) das estirpes, 22 das quais pertencentes ao ribotipo (RT) 017 (apenas toxina B positivo), todas provenientes do mesmo hospital, durante o período em estudo, e com perfil de multiresistência. Pela análise dos dados de sequenciação dos genomas, foram identificadas duas substituições de aminoácidos (Ala555Thr e Tyr721Ser) nos domínios funcionais de duas enzimas envolvidas na síntese do peptidoglicano (penicillin-binding proteins - PBP). Uma PBP adicional foi também identificada nas estirpes RT017. Este estudo descreve pela primeira vez alterações em PBPs como base genética provável da resistência ao imipenemo em C. difficile.Clostridium dif ficile is a major cause of healthcare-associated infections. Here, we characterized C. dif ficile strains isolated in Por tuguese hospitals, in order to search for impenem resistance and the underlying genetic determinants. Imipenem susceptibility testing by agar gradient dif fusion was per formed on 191 C. dif ficile strains, isolated from 15 portuguese hospitals, between 2012-2015. Some of the imipenem-resistant and imipenem-susceptible strains were selected for downstream phenotypic analyses and for whole genome sequencing (WGS). Resistance to imipenem was detected in 24 (12.6 %) strains, 22 of which were ribotype (RT) 017 strains, only positive for toxin B, isolated in the same hospital, and presenting resistance to several other antibiotics. Through analysis of WGS data, two amino acid changes (Ala555Thr and Tyr721Ser) targeting the transpeptidase domain of two penicillin-binding proteins (PBP) were identified. An additional PBP was also identified in this ribotype. We describe, for the first time, mutations in PBP-encoding genes as the probable genetic basis for C. dif ficile imipenem resistance.Este trabalho foi financiado pelo INSA (projeto 2016DDI1284) e pela Fundação para a Ciência e Tecnologia (bolsa de investigação no âmbito do projeto Pest-C/EQB/LA0006/2011; programa IF IF/00268/2013/CP1173/CT0006, a MS; bolsa de doutoramento PD/BD/105738/2014, a ALM).info:eu-repo/semantics/publishedVersio

Genomic Study of a Clostridium difficile Multidrug Resistant Outbreak-Related Clone Reveals Novel Determinants of Resistance

Background:Clostridium difficile infection (CDI) is prevalent in healthcare settings. The emergence of hypervirulent and antibiotic resistant strains has led to an increase in CDI incidence and frequent outbreaks. While the main virulence factors are the TcdA and TcdB toxins, antibiotic resistance is thought to play a key role in the infection by and dissemination of C. difficile.Methods: A CDI outbreak involving 12 patients was detected in a tertiary care hospital, in Lisbon, which extended from January to July, with a peak in February, in 2016. The C. difficile isolates, obtained from anaerobic culture of stool samples, were subjected to antimicrobial susceptibility testing with Etest®strips against 11 antibiotics, determination of toxin genes profile, PCR-ribotyping, multilocus variable-number tandem-repeat analysis (MLVA) and whole genome sequencing (WGS).Results: Of the 12 CDI cases detected, 11 isolates from 11 patients were characterized. All isolates were tcdA-/tcdB+ and belonged to ribotype 017, and showed high level resistance to clindamycin, erythromycin, gentamicin, imipenem, moxifloxacin, rifampicin and tetracycline. The isolates belonged to four genetically related MLVA types, with six isolates forming a clonal cluster. Three outbreak isolates, each from a different MLVA type, were selected for WGS. Bioinformatics analysis showed the presence of several antibiotic resistance determinants, including the Thr82Ile substitution in gyrA, conferring moxifloxacin resistance, the substitutions His502Asn and Arg505Lys in rpoB for rifampicin resistance, the tetM gene, associated with tetracycline resistance, and two genes encoding putative aminoglycoside-modifying enzymes, aadE and aac(6′)-aph(2″). Furthermore, a not previously described 61.3 kb putative mobile element was identified, presenting a mosaic structure and containing the genes ermG, mefA/msrD and vat, associated with macrolide, lincosamide and streptogramins resistance. A substitution found in a class B penicillin-binding protein, Cys721Ser, is thought to contribute to imipenem resistance.Conclusion: We describe an epidemic, tcdA-/tcdB+, multidrug resistant clone of C. difficile from ribotype 017 associated with a hospital outbreak, providing further evidence that the lack of TcdA does not impair the infectious potential of these strains. We identified several determinants of antimicrobial resistance, including new ones located in mobile elements, highlighting the importance of horizontal gene transfer in the pathogenicity and epidemiological success of C. difficile

A Negative Feedback Loop That Limits the Ectopic Activation of a Cell Type–Specific Sporulation Sigma Factor of Bacillus subtilis

Two highly similar RNA polymerase sigma subunits, σF and σG, govern the early and late phases of forespore-specific gene expression during spore differentiation in Bacillus subtilis. σF drives synthesis of σG but the latter only becomes active once engulfment of the forespore by the mother cell is completed, its levels rising quickly due to a positive feedback loop. The mechanisms that prevent premature or ectopic activation of σG while discriminating between σF and σG in the forespore are not fully comprehended. Here, we report that the substitution of an asparagine by a glutamic acid at position 45 of σG (N45E) strongly reduced binding by a previously characterized anti-sigma factor, CsfB (also known as Gin), in vitro, and increased the activity of σG in vivo. The N45E mutation caused the appearance of a sub-population of pre-divisional cells with strong activity of σG. CsfB is normally produced in the forespore, under σF control, but sigGN45E mutant cells also expressed csfB and did so in a σG-dependent manner, autonomously from σF. Thus, a negative feedback loop involving CsfB counteracts the positive feedback loop resulting from ectopic σG activity. N45 is invariant in the homologous position of σG orthologues, whereas its functional equivalent in σF proteins, E39, is highly conserved. While CsfB does not bind to wild-type σF, a E39N substitution in σF resulted in efficient binding of CsfB to σF. Moreover, under certain conditions, the E39N alteration strongly restrains the activity of σF in vivo, in a csfB-dependent manner, and the efficiency of sporulation. Therefore, a single amino residue, N45/E39, is sufficient for the ability of CsfB to discriminate between the two forespore-specific sigma factors in B. subtilis